Characterization of the MATα pre-/pro- peptide by mutagenesis as a means to optimize secretion in pichia pistoris

<p>The unicellular yeast, <em>Pichia pastoris</em> has currently emerged as one of the most popular host systems for heterologous proteins due to its relatively cheap cost, easy genetic manipulability, ability to perform post-translational modifications on proteins, and respiratory growth capabilities which allow it to be cultured in very high concentrations. Over 700 foreign proteins have been recombinantly expressed using <em>P. pastoris</em>.</p> <p>Although <em>P. pastoris</em> appears to be an ideal host system, its main drawback is its inability to efficiently export some heterologous proteins into the extracellular medium. The incorporation of <em>S. cerevisiae</em>'s <em>MAT</em>α <em>pre-pro</em> signal leader (<em>MAT</em>α) has led to increased protein secretion in most cases. <em>MAT</em>α is thus used in the production of 90% of all proteins secreted in <em>P. pastoris</em>. However secretion efficiency still remains a problem.</p> <p>It has been suspected that low secretion may be attributed to improper extracellular targeting (a function of <em>MAT</em>α). In order to address these issues there has been a precedent for performing limited mutagenesis of a signal leader peptide (like <em>MAT</em>α) to increase protein secretion. In one study the insertion of a 10 amino-acid residue into <em>MAT</em>α resulted in a 5-fold increase in secretion of bacterial phytase, an important industrial enzyme. Despite this success there have been no systematic mutagenesis processes which would help elucidate the reason behind this case of increased secretion.</p> <p>In our study, we performed a series of mutagenesis events, both random and site directed, with the intent of illuminating the mechanisms of <em>MAT</em>α that contribute to secretion. As a result were able to create a novel secretion signal (pLL3) with enhanced secretion levels of our reporter protein HRP.</p>

Name thesis:degree_name

Master of Science (M.S.)

Level thesis:degree_level

Thesis - Pacific Access Restricted

Discipline thesis:degree_discipline

Biological Sciences

Year dc:date.available

2009

Author dc:creator

• Kim, Daniel

Contributors dc:contributor

• Geoff Lin-Cereghino

• Proteins
• Synthesis
• Biotechnology
• Biology
• Life Sciences

Licence

• http://rightsstatements.org/vocab/InC/1.0/

Repository record dc:identifier

https://scholarlycommons.pacific.edu/uop_etds/738

OAI identifier oai:identifier

oai:scholarlycommons.pacific.edu:uop_etds-1737